While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM ...(paired guide RNAs for alternative exon removal), a CRISPR-Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of 'poison' cassette exons, which disrupt their host genes' reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.
The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor ...progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3
mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ
/TNFα
/IL10
CD4
and CD8
T cells. Immunodepletion of CD4
but not CD8
T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.
This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists.
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